Bacterial populations have the ability to generate persister cells that can tolerate exposure to lethal doses of antibiotics and persistence may be responsible for relapses seen after standard antibiotic treatments of bacterial infections. Francisella tularensis is a Gram-negative bacterium that causes the deadly disease of tularemia and is commonly spread by ticks in the US. It is highly infectious and is classified as a Tier1 Select agent by the DHHS and the CDC. F. tularensis enters and replicates in the cytoplasm of different host cells including macrophages, overcoming nutritional and other stresses in this environment. Tularemia treatment with standard antibiotic therapy may result in relapse and there is a great need for development of adjunct therapy to combat infection with this pathogen. Understanding the mechanisms that govern persister formation is therefore critical. In the well-studied model organism Escherichia coli, development of persister cells is regulated by the hipBA locus in conjunction with ten other toxin-antitoxin (TA) loci. This proposal has three aims that will test if F. tularensis forms persisters in response to signals received by hipBA in the mammalian intracellular environment. Aim 1 will examine whether the intracellular environment influences persistence. Aim 2 of this proposal will explore the role of the hipBA genes in persistence of F. tularensis. Aim 3 will seek to identify signals and pathways that regulate persister cell formation. Understanding the mechanisms of hipBA- mediated persistence in this organism may lead to identification of targets for development of new adjunct therapies directed at persisters.